To obtain the folding pattern of a protein, the relative location and packing of secondary structure should be determined. A direct approach to obtain these geometric constraints is the measurement of internitroxide distances for selected pairs of sites with each member of a pair preferably on the exposed surface of one of the secondary structures. Two unpaired electrons separated by a distance r are coupled through the distance-dependent electron-electron dipole interaction. Theoretical and experimental approaches have been developed for the analysis and use of this interaction. Thus the stage is set to evaluate the possibility of obtaining folding patterns using SDSL. Relevant questions include the uniqueness of the obtained structure, its resolution, and the influence of the nitroxide side chain flexibility. For this purpose, we are using a protein model system with accurately known X-ray structure, T4 lysozyme. Sequential cysteine mutants were constructed in the C-terminal domain spanning the region between residues 80 and 163. The helical bundle in this domain consists of five principal helices arranged in a pattern similar to that used in the packing of membrane proteins. Based on the secondary structure assignment obtained from SDSL, double cysteine mutants were constructed. Preliminary results indicate that the obtained secondary structure and topography are entirely consistent with the X-ray structure. We are currently investigating the number of distance constraints necessary to generate a structure at a biologically relevant resolution.